PROJECT SUMMARY/ABSTRACT Heterotopic ossification (HO), the formation of ectopic endochondral bone in skeletal muscle and soft tissues, is a significant cause of morbidity from joint immobility and pain. The precise mechanisms responsible for HO are not known; however, its association with trauma, inflammation and biomechanical stress suggests a process of disordered injury repair and homeostasis. We have explored the underlying mechanisms of a monogenic cause of HO, fibrodysplasia ossificans progressiva (FOP), caused by activating mutations of the bone morphogenetic protein (BMP) type I receptor ALK2, whereas trauma-induced HO appears to be regulated by ALK2, ALK3 and potentially ALK6. FOP and acquired forms of HO share a common mechanism of inappropriate BMP signaling, but the manner by which BMP signals are interpreted to regulate ossification versus tissue regeneration remain incompletely understood. Significant gaps exist in how combinatorial BMP/TGFb signal transduction specifies diverse functions and cell fates in multipotent lineages. To address these mechanistic gaps, an innovative chemical biology platform has been devised using human- derived MSC that have been edited by CRISPR/Cas9 techniques, combined with a novel BMP/TGF-b pharmacologic probe identified from an active NCATS-TRND collaboration. This platform will permit the unequivocal mapping of individual ligand and receptor signaling, and the downstream impact on MSC plasticity in a non-overexpressed human cell system. This assay provides a platform for a high throughput screen to be performed with collaborators at NCATS-TRND to identify mechanistically novel modulators of ALK2, ALK3 and ALK6. Finally, insights and candidate molecules identified from these studies will be validated in a conditional knock-in mouse model of FOP, and in an authentic mouse model of trauma- and inflammation-induced HO. These studies will provide critical tools and insights into how the BMP signaling pathway contributes to pathologic tissue remodeling in musculoskeletal and degenerative disease, as well as in a broader set of conditions in which HO is associated with senescence, inflammation, and metabolic stress. New assay technologies, tool compounds, and translatable insights will be produced as a result of this work that will be relevant to many other disease-oriented fields of study.